Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We examined alterations in cell morphology and expression of adhesion molecules in response to a general protein kinase inhibitor K252a treatment of non-adherent colon adenocarcinoma Colo201 cells. K252a induced rapid cell adhesion and spreading with concomitant formation of actin stress fibers. A protein kinase A inhibitor KT5720 also induced cell adhesion, but the rate of spread was slower than that seen with K252a. These adhesions were mediated by integrin molecules since cell adhesion required Mg2+, Mn2+ or Ca2+, and was inhibited by monoclonal antibodies for integrins alpha2 and beta1. Indirect immunofluorescence microscopic observations revealed that integrin alpha2 and beta1 molecules in K252a-treated cells were concentrated at sites of focal adhesion, but expressions of integrin molecules were not modulated. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin increased during K252a- or KT5720-induced cell adhesion. Immunosuppressants FK506 and cyclosporin A suppressed the K252a-induced cell adhesion and abolished tyrosine phosphorylation of cellular proteins including FAK and paxillin. Furthermore, W7 and calmidazolium, inhibitors of calmodulin, also inhibited the cell adhesion. Based on findings that FK506 and cyclosporin A are inhibitors of the calcium calmodulin-dependent protein phosphatase, calcineurin, this phosphatase may regulate integrin-dependent cell adhesion and spread of Colo201 cells. This Colo201 cell model provides a pertinent system for studying molecules involved in signal transduction pathways and can shed light on mechanisms of metastasis and invasion of colon carcinoma cells.
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PMID:Rapid adhesion and spread of non-adherent colon cancer Colo201 cells induced by the protein kinase inhibitors, K252a and KT5720 and suppression of the adhesion by the immunosuppressants FK506 and cyclosporin A. 987 66

PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. Finally, we have found that PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and have detected clusters of PINCH that are colocalized with the alpha5beta1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways.
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PMID:The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells. 1002 29

Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc.
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PMID:Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc. 1018 52

Recently, we demonstrated that pulsatile mechanical stretch induced rapid secretion of vascular endothelial growth factor (VEGF) by cultured rat cardiac myocytes in vitro. To investigate whether pulsatile stretch activates intracellular signaling in cardiac myocytes, we examined the activation of mitogen-activated protein kinase (MAPK) family members and focal adhesion kinase (p125(FAK)) in cultured rat cardiac myocytes. We found that pulsatile stretch rapidly phosphorylated p44/p42 MAPKs (extracellular signal-regulated protein kinase [ERK] 1/2), stress-activated protein kinase (SAPK), p38MAPK, and p125(FAK). The stretch-induced activation of ERKs was at least partly mediated by VEGF, which was shown to be induced by transforming growth factor (TGF)-beta, and was also partly dependent on tyrosine kinases as well as protein kinase C (PKC). These data provide the direct evidence that pulsatile stretch can activate intracellular signaling in cardiac myocytes and that this was at least partly mediated by VEGF, which may play a role in cardiac adaptation to mechanical overload.
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PMID:Pulsatile stretch activates mitogen-activated protein kinase (MAPK) family members and focal adhesion kinase (p125(FAK)) in cultured rat cardiac myocytes. 1033 7

HEF1 is a recently described p130(Cas)-like docking protein that contains one SH3 domain and multiple SH2 binding motifs. In B cells, HEF1 is phosphorylated by a cytoskeleton-dependent mechanism that is triggered by integrin ligation. However, the induction of HEF1 phosphorylation by G protein-coupled receptors has not been reported. We found that HEF1, but not p130(Cas), is tyrosine-phosphorylated following stimulation of the rabbit C1a calcitonin receptor stably expressed in HEK-293 cells. The calcitonin-induced tyrosine phosphorylation of HEF1 increased in a time- and dose-dependent manner. Dibutyryl cAMP and forskolin had little or no effect on HEF1 phosphorylation, and the protein kinase A inhibitor H89 failed to detectably inhibit the response to calcitonin, indicating that the G(s)/cAMP/protein kinase A pathway does not mediate the calcitonin effect. Pertussis toxin, which selectively blocks G(i/o) signaling, also had no effect. Increasing cytosolic Ca(2+) with ionomycin stimulated HEF1 phosphorylation and preventing any calcitonin-induced change in cytosolic calcium by a combination of BAPTA and extracellular EGTA completely blocked the calcitonin-induced tyrosine phosphorylation of HEF1. Phorbol 12-myristate 13-acetate also induced HEF1 tyrosine phosphorylation, and the protein kinase C inhibitor calphostin C completely inhibited both calcitonin- and phorbol 12-myristate 13-acetate-stimulated HEF1 phosphorylation. Calcitonin also induced the tyrosine phosphorylation of paxillin and focal adhesion kinase, and the association of these two proteins with HEF1. Pretreatment with cytochalasin D, which disrupts actin microfilaments, prevented the calcitonin-induced HEF1 and paxillin phosphorylation. In conclusion, the calcitonin-stimulated tyrosine phosphorylation of HEF1 is mediated by calcium- and protein kinase C-dependent mechanisms and requires the integrity of the actin cytoskeleton.
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PMID:Cytoskeleton-dependent tyrosine phosphorylation of the p130(Cas) family member HEF1 downstream of the G protein-coupled calcitonin receptor. Calcitonin induces the association of HEF1, paxillin, and focal adhesion kinase. 1045 89

Apoptosis has been shown to be involved in endocrine tissue homeostasis as well as regression due to hormone deprivation. The goal of this study was to induce apoptosis and to investigate a potential role of TSH as a survival factor in thyroid follicular cells (FRTL-5) in vitro. Our results indicated that FRTL-5 cells underwent anchorage-dependent apoptosis when plated in the absence of serum and hormones, but when the cells became attached to the substrate by addition of TSH in the medium, apoptosis was prevented. The apoptosis was evaluated by positive terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling staining, typical apoptotic bodies by electron microscopy, DNA ladder by gel electrophoresis, and subdiploidy by propidium iodide-stained flow cytometry. TSH was shown to prevent apoptosis and maintain cell viability. cAMP partly mimicked this effect, which was inhibited by a specific inhibitor of protein kinase A, H-89. While investigating the mechanisms of apoptosis, we observed that the phosphorylated focal adhesion kinase was strengthened by TSH. Furthermore, FRTL-5 cells were found to undergo growth arrest in the G1 phase in the absence of TSH, accompanied by an elevated level of cyclin-dependent kinase inhibitor, p27, and a decreased level of cyclin D. In contrast, TSH promoted transition from G1 to S phase by decreasing P27 protein and increasing cyclin D expression. We concluded that in addition to regulating growth and differentiation, TSH may function as a survival factor in thyroid cells by preventing anchorage-dependent apoptosis in FRTL-5 cells partly via the cAMP pathway.
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PMID:Thyrotropin prevents apoptosis by promoting cell adhesion and cell cycle progression in FRTL-5 cells. 1057 64

In this study we investigated the signaling event induced by adhesion of human trabecular meshwork (TM) cells to extracellular matrix (ECM) elements such as fibronectin. The role of tyrosine phosphorylation in adhesion was evaluated. A number of intracellular entities involved in the adhesion-mediated pathways were identified. For the experiments, human TM cells were seeded onto fibronectin- or polylysine (negative control)-coated plates. Fifteen, 30, 90 and 240 min after the seeding, cell lysates were collected. Immunoblotting analysis revealed that tyrosine phosphorylation occurred within 15 min of adhesion of TM cells to fibronectin and the level increased with time. The phosphotyrosyl proteins had molecular masses 25-220 kDa. A much lower level of tyrosine phosphorylation was observed when cells were plated on polylysine. Immunoprecipitation experiments indicated that the phosphotyrosine-containing proteins included focal adhesion kinase, paxillin, phosphatidylinositol 3-kinase and mitogen activated protein kinase. Within 30 min of adherence to fibronectin, human TM cells immunostained for paxillin and phosphotyrosine and exhibited prominent focal contacts. When treated with tyrosine kinase inhibitors genistein and herbimycin A and a protein kinase C (PKC) pseudosubstrate peptide inhibitor, cell adhesion to fibronectin was compromised and focal contact formation was limited. These results demonstrated that in human TM cells, tyrosine kinase was activated upon their adherence to fibronectin. PKC also appeared to play a role in modulation of the cell-matrix adhesion process. The current study provides insight into the signaling pathways that are linked to the ECM-induced events in TM cells. Elucidation of the hierarchy of signal responses may help develop strategies manipulating the cell-matrix interactions in the TM system.
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PMID:Signal transduction mediated by adhesion of human trabecular meshwork cells to extracellular matrix. 1086 94

HEF1 (human enhancer of filamentation 1) is a member of a docking protein family that includes p130(Cas) and Efs. Through assembly of multiple protein interactions at focal adhesion sites, these proteins activate signaling cascades in response to integrin receptor binding of the extracellular matrix. The HEF1 protein is cell cycle regulated, with full-length forms cleaved in mitosis at a caspase consensus site to generate an amino-terminal 55-kDa form that localizes to the mitotic spindle. The identification of a caspase cleavage site in HEF1 led us to investigate whether HEF1 belongs to a select group of caspase substrates cleaved in apoptosis to promote the morphological changes characteristic of programmed cell death. Significantly, inducing expression of HEF1 in MCF-7 or HeLa cells causes extensive apoptosis, as assessed by multiple criteria. Endogenous HEF1 is cleaved into 65- and 55-kDa fragments and a newly detected 28-kDa form in response to the induction of apoptosis, paralleling cleavage of poly(ADP-ribose) polymerase and focal adhesion kinase (FAK); the death-promoting activity of over-expressed HEF1 is associated with production of the 28-kDa form. While the generation of the cleaved HEF1 forms is caspase dependent, the accumulation of HEF1 forms is further regulated by the proteasome, as the proteasome inhibitors N-acetyl-L-leucinyl-L-leucinyl-L-norleucinyl and lactacystin enhance their stability. Finally, the induction of HEF1 expression also increases Jun N-terminal protein kinase (JNK) activation, and activated JNK colocalizes with HEF1, implicating this pathway in HEF1 action. Based on these results, we propose that dysregulation of HEF1 and its family members along with FAK may signal the destruction of focal adhesion sites and regulate the onset of apoptosis.
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PMID:The docking protein HEF1 is an apoptotic mediator at focal adhesion sites. 1086 74

Thrombin-induced endothelial monolayer hyperpermeability is thought to result from increased F-actin stress fiber-related contractile tension, a process regulated by the small GTP-binding protein Rho. We tested whether this process was dependent on the Rho-associated protein kinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects of Y-27632 on thrombin-induced myosin light chain phosphorylation (MLCP) and tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) and paxillin were measured by Western blotting. F-actin organization and content were analyzed by digital imaging, and endothelial monolayer permeability was measured in bovine pulmonary artery endothelial cell (EC) monolayers using a size-selective permeability assay. Y-27632 enhanced EC monolayer barrier function due to a decline in small-pore number that was associated with increased EC surface area, reduced F-actin content, and reorganization of F-actin to beta-catenin-containing cell-cell adherens junctions. Although Y-27632 prevented thrombin-induced MLCP, stress fiber formation, and the increased phosphotyrosine content of paxillin and p125(FAK), it attenuated but did not prevent the thrombin-induced formation of large paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to be partially ROCK dependent.
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PMID:ROCK mediates thrombin's endothelial barrier dysfunction. 1089 31

Migration of rat ascites hepatoma (MM1) cells, invasion and phagokinetic movement were induced by the combination of lysophosphatidic acid (LPA) and fibronectin (FN). Induction of migratory activity was tightly correlated with morphological change of MM1 cells from spherical or polygonal-shaped cells to fusiform-shaped ones with pseudopodia. MM1 cells were mobile in a fusiform shape, whereas those of a spherical or polygonal shape were not. A small GTPase Rho and one of its downstream effectors ROCK (Rho-associated coiled-coil forming protein kinase), play essential roles in these processes, as evidenced by suppression of migration and morphological change of MM1 cells by Clostridium botulinum C3 exoenzyme, an inhibitor of Rho, or by Y-27632, an inhibitor of ROCK. Y-27632 also suppressed the formation of fusiform-shaped pseudopodia-carrying MM1 cells that was induced by stimulation with the combination of LPA and FN. LPA and FN also evoked the formation of focal adhesions and actin bundles, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. The inhibitory effect of Y-27632 on LPA-induced migration and morphological change of MM1 cells was considered to be mediated, at least in part, by impaired formation of focal adhesions and actin bundles. Y-27632 suppressed LPA-induced tyrosine phosphorylation of FAK and paxillin, suggesting that ROCK regulates these molecules and Y-27632 inhibits cellular migration and morphological change, at least in part, through this regulation.
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PMID:Y-27632, an inhibitor of rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase. 1096 22


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